designed a new set of universal primers for vertebrate mitogenomes
by referring its most conservative regions and then amplify mito-
genomes, and its PCR product was used to make probe. Second, we
prepared 2 kb length of libraries. Third, the libraries and probe
were mixed to enrich target mtDNA. Then we sheared the enriched
long-range library to approximately 300–700 bp and followed
downstream library construction steps for sequencing. In the
downstream sequence experiment, an Ion Torrent Personal
Genome Machine (PGM) was used to sequence because it is fast
and relatively inexpensive in terms of each run (not price per base).
Each run using 316 chip generated over 800 Mb for 60 samples,
and the data size for each sample is more than 10 Mb in general.
These generated data was sufficient for de novo assembly of a
complete mitochondrial genome. Although we sequenced with
Ion Torrent platform, the protocol could also be applied to
Fig. 1Schematic pipeline for enriching mitochondrial DNA to high-throughput
sequencing. The green line represents using the pair of long-range PCR
(LR-PCR) amplicons to directly construct a library. Compared to standard
library, the long-range library capture hybridization strategy (LR-LCR) has
modification in library construction 1 and 2. LR-LCR requires a long
fragment during shearing in library construction 1 for capturing high variable
loci and additional library construction 2 for PGM sequencing. In standard
hybridization, there is no construction library 2 and the enriched fragments
directly sequenced
Capture Hybridization of Long-Range DNA Fragments for High-Throughput Sequencing 33